Process for producing rifamycin sv

ABSTRACT

A fermentation process for preparing rifamycin SV employing a heretofore unknown microorganism is described, said microorganism being classified as a member of the genus Micromonospora and being herein named, Micromonospora ellipsospora 71372.

United States Patent [1 1 Weinstein et al.

[ Aug. 26, 1975 [73] Assignee: Schering Corporation, Kenilworth,

[22] Filed: May 15, 1974 [21] Appl. No.: 469,972

[52] US. Cl. 195/96 [5 1] Int. Cl. C12D 9/20 [58] Field of Search 195/96, 80 R [56] References Cited UNITED STATES PATENTS 3,597,324 8/[971 Lancini et al 195/80 R Primary ExaminerAlvin E. Tanenholtz Altorney, Agenl, or FirmCarver C. Joyner; Stephen B. Coan; Raymond A, McDonald [57] ABSTRACT A fermentation process for preparing rifamycin SV employing a heretofore unknown microorganism is described, said microorganism being classified as a member of the genus Micromonospora and being herein named, Micmmonospora ellipsospora 7l372.

5 Claims, No Drawings PROCESS FOR PRODUCING RIFAMYCIN SV This invention relates to a new process for preparing a known antibiotic. More particularly. this invention relates to a process for preparing rifamycin SV by fermenting strains of Micmnmuni-pom ellipxosporu under controlled conditions.

BACKGROUND OF THE lN\'ENTlON 1U Rifamycin S\' is a known antibiotic whose preparation via chemical means from substances produced by fermenting strains of Slrepmnrvcev medilerranei has been described. In a recent patent (US. Pat No.

3.597.324. issued Aug. 3. 197i is described a method for producing the antibiotic via microbiological means using a mutant strain of S. mediterrunez'.

We have discovered that Mic'mmmmspuru cllipsospnra 71372 produces rifamycin SV when fermented under submerged aerobic conditions in the presence of assimilable sources of carbon and nitrogen.

The Microorganism Mit-rmnmwspuru ellipsoxpuru 71372 (sometimes herein designated as M. ellipsospum) was classified as a 3 species of the genus Micromonospora on the basis of whole cell analysisv And on the basis of growth and physiological properties it was determined to be a new species. Whole cell analysis revealed that the microorganism contained meso-diaminopimclic acid (DAP).

2 Xylose and arabinose as characteristic chemical components. The microorganism is also unique in that it is the only species of the genus Micromonospora known in the art which is reported to produce rifamycin S\'.

Representative samples of M. cllipruspnru 7137] have been deposited with the ARS Culture Collection Investigations Fermentation Laboratory. Northern L'ti' lization Research and Development Division. Peoria. Illinois 6 l 604 where it was assigned the ascension number NRRL 802 l. A significant characteristic of the microorganism is its ability to elaborate the antibiotic. rifamycin S\'. Thus. this invention relates to a process for producing rifamyein S\' by Mir'rommiorpum (Nipsosporu 7l372-NRR1. XUZI or by mutants or variants thereof having the characteristics of NRRl. 8021 as set forth in the tables below:

In describing the color formations the following system and references are employed: The color designation consists oftwo designates. The first is a color name taken from the Descriptive Color Name Dictionary." by Taylor. Knoche and Granville. published by the Container Corporation of America. 1950 (L'.S.A.). with a color chip number corresponding to the color name; said chip number taken from the "Color Har mony Manual." 4th edition. I958. published by the Container Corporation of America. U.S.A. The second designate. consists of a eolonnantc and number which refers to the synonym or near synonym found in the National Bureau of Standards. Circular 553. Nov. 1.

1955 (L.S.A.).

Table l (irovvth Characteristics on Various Media Surface tc tture Aerial Soluble Comments (ironth of and form... mycelium Surface color tdifl'usible) Physiological Icultul'c colony tvigor) plicate. viscid. present. of colony. chip pigment by properties on name or -Ht- 1 flat. furrowed. absent. number and NBS transmitted particular number) good fair poor glabrous. powdery other equivalent light substrate file number Czapek -l+-lplicatc absent glpn dark slight Sucrose brown. dark l'lltl\\lll\h Agar olive [OX tint (ilucosc flat. often furabsent g4pg dark lug none Asp-.iragine rowed or granw gage tan: Agar lar somewhat strong broun 55 moist Calcium raised. absent g4pe orange none Malate granular rust; deep Agar orange 5t C/apck l'loculeni absent g3ic Ll. amber none (ilucose no surface dark orange Solution growth yellow 72 Ordinary Agar insufficient for (Water agarl characterization Nutrient to I flat absent g3ic Lt. amber none Agar dark orange yellow 73 foefflcr's +H' plicate absent gSpg rust reddish substrate Serum brown. strong bro a partially Medium brown 55 decomposed ('alcium flat. sometimes abscnt g-ipc russet none Citrate \gar furroited orange; strong orange 51) g-iia Apricot. Light bro n Potato -CaCt') plicate absent Strong Orange very light Medium 50 to g-lpi Oak brown CaCO:, plicate absent Bronn; Strong Brovm 55 g-tpe Orange Rust. Deep Orange 5| P ptono t insufficient (ilucose Ioi description Medium Iigg Agar plicatc absent g-lle 'l urf tan; none substrate Medium Light brovin 7 not decomposed g-tpe Orange (ielatin t flat absent Rust. Deep none Mcdlum Orange il Starch flat absent gipi Copper i Bronn. Strong taint brown Table lV-continued Carbohydrate ['tilizauon solvent. By evaporating the extracts to a residue in vacuo. dissolving the residue in acetone and precipitating with ethyl ether an orange-yellow solid is obtained. Chromatographic comparison of the product with rifamycins O. B. S and SV using Whatnian No. I paper and a solvent system composed ofn-amyl alcoholzn-butanol (91l saturated with pH 8.6 phosphate buffer is as set forth in Table VI below.

By the above-described chromatographic system. rifamycin SV and rifamycin S are indistinguishable. However. by subjecting the product to oxidation. followed by thin layer chromatography and bio-autography. it can be unequivocally determined that the prod act of the fermentation is in fact rifamycin SV. TABLE mele/itose It-l)'l ;|lllLltl\C l)l+l I ral'finose Dr. H glucose H Ll+l t ldcstrose) rliamnose l ositi\c L'ontrol glycerol I Dt r ribose l ll\ll0l t salient t ii-lactose sucrose 1- t l)l-l le\ulose H Dll'l [fructose xy lose Negative [)t *1 lnannitol Control No Carbohydrate (ell .tll analysis {diagnostic cltenueal L'Ullsllltlllllhl lype |l mlcromonospora type meso l).\l Aftlhllluse t \lose Table V Physiological Properties (iroytth Response Maximal Minimal ()ptimal pH iii 75 Temperature 4llC 28% Oxygen requirements. v 3S aerobic. microaerophilic. anaerobic. Hydrolysis reactions (iood Fair Poor (ielatin (liquelactionl Starch -H+ Milk tpeptonization) Cellulose (decomposition) 4-H- 10 'lyrosine (dissolving of t crystals) tmelanin formation) 1 Miscellaneous Chemical Reactions Positive Variable Negative Nitrate Reduction I/ The Fermentation A sample of M. ellipsospuru 71 372 from an agar slant is used to inoculate a 300 ml flask containing 100 ml of a suitable medium. such as the one set forth below. The flask is incubated for from about 40 to about 72 hours at from about to about 38C. preferably about C. The incubation is performed with continuous agitation at from about 250 to about 300 rpm. The inoculum is then frozen and maintained in a frozen state until ready for use. a 571 v/v inoculum being usually employed. ln general. the fermentation involves one or more germination stages and one production stage. Germination is advantageously conducted at about 28C commencing at about pH 7.5 and continuing for about 72 hours with agitationv A 571 inoculum is used to inoculate a second germination stage or to inoculate the production stage. Peak antibiotic production is usually attained in from about 96 to I20 hours at from about 25 to about 38C. preferably about 28C with agitation and aeration. Peak antibiotic production is determined by disc dilution test against Slap/l \/()('()((HS ULlrL'llA' ATCC 6538P. Typical germination and production media are as set forth in the specific examples.

Antibiotic lsolation When peak antibiotic production is attained. the antibiotic is harvested by methods generally used in the art. Extraction of the fermentation medium with a nonwater miscible polar organic solvent may advantageously be employed. ethyl acetate being the preferred Rf as shown by bioautography against S. aureus ATCC 6538? Fermentation Product Rifamyein B S S\ 0.87 (1.40 0.40 [L87 .87

EXAM PLE l Fermentation of Micromonospora Ellipsospora 7 l 372 A. Germination Stage Transfer under aseptic conditions 5 ml. of frozen whole broth of M. ellipsospora 7 l 372 to a group of 300 ml. shake flasks containing ml. of sterile medium having the following composition:

Starch Yeast extract Destrose Beef extract 'l ryptose (alcium carbonate Tap water The pH is adjusted to 7.5 with sodium hydroxide prior to sterilization.

lncubatc the flasks and their contents for 2-3 days at about 35C on rotary shakers (25()3()() rpm. 2 inches stroke) and obtain thereby inoeula having luxuriant growth.

8. Fermentation Stage Transfer under aseptic conditions 500 ml of inoculum prepared as described in step A to l4 liter fermentors containing liters ofsterile medium A or medium B having the following compositions:

Production Media 'llalllmore Biological Lat -'|)||'co Laboratories The pH is adjusted to 7.5 with sodium hydroxide prior to sterilization.

Conduct the fermentation at about 28C. pH 6.87.3. with stirring at about 350 rpm. aeration at about 3.5 liters per minute for about 90 hours then commence sampling the fermentation. Submit the samples to disc dilution tests against SruplryIumccs uureus 65351 to determine when peak antibiotic production is attained then isolate the antibiotic promptly.

EXAMPLE 2 Isolation of Rifamycin SV Extract the whole broth from Example I repeatedly with liter volumes of ethyl acetate until antibiotic is completely removed (Le. a 100 fold concentrate of extract shows no u.v. absorption at 223 mp. 314 mu or 445 mu). Combine the extracts and concentrate in vacuo to a residue. dissolve the residue in acetone and precipitate by pouring into 10 volumes of ethyl ether with stirring. Remove the orange-yellow precipitate by filtration and dry at 40C in vacuo.

Yield 0.53 g

Chromatography of the precipitate on thin layer silica gel plates using a solvent system consisting of chloroform: methanol (9:1 followed by bioautography against Slaphylocm'cus aureus ATCC 6538? indicates the presence of one biologically active compound which remains at the origin of the plate (i.e. R;=0.00). Authentic rifamycin SV has the same R,.

EXAMPLE 3 Conversion of Rifamycin S\' to Rifamyein S Dissolve the precipitate obtained in Example 2 (0.5 g) in acetone with stirring and add a solution of potassiuin ferricyanide (3 equivalents). Stir the resulting mixture for 1 hour at room temperature. then add 5 volumes of water. Extract the rifainycin S with ethyl acetate. concentrate the extract to a residue and obtain thereby a red solid which on the abovc-described solvent systcm has an R, of 0.73. The product is indistinguishable from rifamycin S by physicochemical and biological evaluation.

The LR. spectrum of the ritamycin S produced in Example 3 shows the following absorption bands:

Wavelength microns 2.95 (m.brd.l 8.63 tnrhrd.) 3.40-3.50 INujol) 8.95 (W) 3.75 (.brd.) 4.20 (w) 5.84 tshl 9.45 tnLbrd.) 6.05 (s.brd.) 10.27 (|n.brd.i (1.27 (W! 10.57 (wt h,-l 5 lsh.) 10.95 (W) 6.52 )s.hrd.) ll.lR tw) (1.83 (Nujol) 12.43 (w) 7.25 (Mujoll 12.75 (wt 7.931s] 13.15 t) 8.07 (\sbrdl 138R (Nujoll Notations: \x -vcr strong. -slrong. m rttcdiunl. cal brd-broad. sh' houldcr We claim:

I. A process for preparing rifamycin SV which comprises cultivating Mivmmvmls mru ei'llipsospora 71372. having the identifying characteristics of NRRL 8021. and the rifamyein SV producing mutants and variants thereof. in an aqueous nutrient medium under aerobic conditions until substantial antibiotic activity is imparted to the medium and isolating rifamycin SV from said medium.

2. A process according to claim I wherein the nutrient medium contains an assimilable source of carbon and nitrogen.

3. A process ot'claim 1 wherein Micromrmnsporu e!- Iipsospora 71372 NRRL 8021 is cultivated.

4. A process according to claim 2 wherein Miz'romo- "()SPOI'U ellipsos mra 71372 is cultivated at a temperature from about 25C to about 38C for from about 96 to about hours and at a pH of from about 6.5 to about 7.5.

5. A process according to claim 1 wherein rifamycin SV is extracted from the medium with a water immiscible polar organic solvent and subsequently isolated 

1. A PROCESS FOR PREPARING RIFAMYCIN SV WHICH COMPRISES CULTIVATING MICROMONOSPORA ELLIPSOSPORA 71372, HAVING THE IDENTIFYING CHARACCTERISTICS OF NRRL 8021, AND THE RIFAMYCIN SV PRODUCING MUTANTS AND VARIANTS THEREOF, IN AN AQUEOUS NUTRIENT MEDIUM UNDER AEROBIC CONDITIONS UNTIL SUBSTANTIAL ANTIBIOTIC ACTIVITY IS IMPARTED TO THE MEDIUM AND ISOLATING RIFAMYCIN SV FROM SAID MEDIUM.
 2. A process according to claim 1 wherein the nutrient medium contains an assimilable source of carbon and nitrogen.
 3. A process of claim 1 wherein Micromonospora ellipsospora 71372 NRRL 8021 is cultivated.
 4. A process according to claim 2 wherein Micromonospora ellipsospora 71372 is cultivated at a temperature from about 25*C to about 38*C for from about 96 to about 120 hours and at a pH of from about 6.5 to about 7.5.
 5. A process according to claim 1 wherein rifamycin SV is extracted from the medium with a water immiscible polar organic solvent and subsequently isolated therefrom. 